Project Summary Antibodies (Abs) are a diverse family of proteins expressed by B cells, and are critical components of the adaptive immune system. They are encoded by hundreds of genes at three primary immunoglobulin (IG) loci. The IG heavy chain locus (IGH), specifically, has been demonstrated to exhibit extreme genetic variability at both the individual and population levels. This IGHV polymorphism provides a rich, albeit understudied, source of humoral immune diversity that may greatly impact host responses to infection and vaccination. One important example is the IGHV1-69 germline gene where the biased use of alleles that encode the critical CDR-H2 Phe54 (F-alleles) to make broadly neutralizing antibodies to the Group 1 influenza A hemagglutinin stem domain (sBnAbs) has been clearly established. Individuals that are homozygous for the Leu54 (L-alleles) rarely can mutate their IGHV1-69 Abs at position 54 and have lower serum sBnAbs titers to this stem domain. Therefore, homozygous LL individuals, who constitute up to 20% of the population, must use other germline genes to mount this sBnAb response. This is an important observation as the lead ?universal? influenza vaccine candidates that are moving into the clinic are directed to the HA stem domain. Toward our goal of investigating the role of Ig polymorphism in mounting a sBnAb response toward the HA stem of Group 1 and 2 influenza A viruses, we have assembled a cohort of 153 healthy volunteers that are part of a seasonal influenza (quadrivalent HA) vaccination study and performed serologic studies for heterosubtypic antibodies to influenza A and B. We have preliminary data that biased use of IGHV1-69 germline gene is not seen for sBnAbs against Group 2 HA and other IGHV germline gene biases have not been explored. We also have genotyped all of the donors at the IGHV1-69 locus and performed expressed antibody repertoire analysis (RepSeq) at three-time points (day 0, 7 and 30) on 20 donors each of FF, FL and LL genotypes. For this R21 proposal we seek to address a fundamental question in host immunity and vaccine responsiveness, is there an important role of IGHV polymorphism in the sBnAb response to the HA stem Group 1 and 2 influenza A viruses. Does this IGHV germline bias affect the quantity and quality of the sBnAb response. For these pilot studies 40 donors will be studied, 20 FF and 20 LL. For Aim 1 we propose to use DropSeq to recover native VH:VL pairing of single memory B (mB cells) that will be coupled with Ab-yeast display and FACS sorting against headless Group 1 and Group 2 HA proteins to identify IGHV usage biases within the FF and LL IGHV1-69 genotypes. We will also determine if ?public? molecular signatures are present as evidenced by pattern of SHM profiles and V(D)J recombinations. For Aim 2, we will perform IgSeq on serum antibodies from pre- and 30 day post vaccination that have been eluted from headless Group 1 and 2 HA affinity columns to examine the impact of IGHV germline gene polymorphism on the serum Ab responses to HA stem. This project will not only yield insights into the role of IGH polymorphism in host defense but will allow us to better understand the barriers in achieving durable immunity to influenza A viruses.